Chemical Composition, Larvicidal and Molluscicidal Activity of Essential Oils of Six Guava Cultivars Grown in Vietnam

Diseases transmitted by mosquitoes and snails cause a large burden of disease in less developed countries, especially those with low-income levels. An approach to control vectors and intermediate hosts based on readily available essential oils, which are friendly to the environment and human health, may be an effective solution for disease control. Guava is a fruit tree grown on a large scale in many countries in the tropics, an area heavily affected by tropical diseases transmitted by mosquitoes and snails. Previous studies have reported that the extracted essential oils of guava cultivars have high yields, possess different chemotypes, and exhibit toxicity to different insect species. Therefore, this study was carried out with the aim of studying the chemical composition and pesticide activities of six cultivars of guava grown on a large scale in Vietnam. The essential oils were extracted by hydrodistillation using a Clevenger-type apparatus for 6 h. The components of the essential oils were determined using gas-chromatography–mass-spectrometry (GC-MS) analysis. Test methods for pesticide activities were performed in accordance with WHO guidelines and modifications. Essential oil samples from Vietnam fell into two composition-based clusters, one of (E)-β-caryophyllene and the other of limonene/(E)-β-caryophyllene. The essential oils PG03 and PG05 show promise as environmentally friendly pesticides when used to control Aedes mosquito larvae with values of 24 h LC50-aegypti of 0.96 and 0.40 µg/mL while 24 h LC50-albopictus of 0.50 and 0.42 µg/mL. These two essential oils showed selective toxicity against Aedes mosquito larvae and were safe against the non-target organism Anisops bouvieri. Other essential oils may be considered as molluscicides against Physa acuta (48 h LC50 of 4.10 to 5.00 µg/mL) and Indoplanorbis exustus (48 h LC50 of 3.85 to 7.71 µg/mL) and with less toxicity to A. bouvieri.


Introduction
Guava (Psidium guajava L.) is a widely grown fruit tree around the world, especially in tropical and subtropical countries such as Pakistan, Mexico, Indonesia, Brazil, Bangladesh, Philippines [1,2], and Vietnam [3]. Parts such as leaves and roots of guava have been used in traditional medicine in many countries, such as Vietnam and China. Guava has been reported to have many beneficial pharmacological effects, such as diabetes, cardiovascular diseases, and cancer [4][5][6].
research was to evaluate the yields, chemical compositions, and larvicidal and molluscicidal activities of six commercially grown guava cultivars in Vietnam. Furthermore, the toxicity of the essential oils to a non-target organism was also evaluated.

Chemical Profiles of Essential Oils
The yield and main components (>4.0%) of essential oils of guava cultivars from Vietnam ranged from 0.4 to 0.53% (v/w) ( Table 1); previous reports showed that essential oils of guava cultivars ranged from 0.11 to 0.9% (v/w) [40,41]. The full analytical results of six guava cultivars are available in the Supplementary Materials Table S1.

Larvicidal Activities
The essential oils of the guava cultivars have been evaluated for larvicidal activity against Ae. aegypti (Tables 2 and 3; major components summarized in Table 4), Ae. albopictus (Tables 5 and 6; major components summarized in Table 7), and Cx. fuscocephala ( Table 8). The essential oils pink flesh smooth skin guava (PG03) and Taiwan guava (PG05) were classified as "exceptionally active" against larvae of all three mosquito species with 24 h LC 50 values < 10 µg/mL [42]. In addition, the essential oils pink flesh rough skin guava (PG04) and Queen guava (PG06) were shown to be "exceptionally active" against larvae of Ae. aegypti with 24 h LC 50 values of 2.71 and 8.51 10 µg/mL, respectively [42]. Two essential oils, pink pearl guava (PG01) and white flesh guava (PG02), were shown to be "very active" against all three mosquito species [42]. Table 2. Larvicidal activity of guava cultivars' essential oils against Aedes aegypti (µg/mL) (Protocol 1).

Material
LC 50

Molluscicidal Activities
The molluscicidal activity of the six guava species did not follow the same trend as the larvicidal activity, i.e., the toxicity to each species of the essential oils was not significantly different, or the difference was not very large. The LC 90 values of the essential oils at 48 h and 72 h were in the range of 6.65-10.54 and 5.04-8.12 µg/mL to P. acuta ( Table 9, molluscicidal activities of major components summarized in Table 10); a range of 3.52-7.71 and 3.02-5.22 µg/mL to I. exustus (Table 11, molluscicidal activities of major components summarized in Table 12), respectively. There were no significant differences in molluscicidal activity between the essential oils. Based on the classification for the plant-based molluscicides, these essential oils were determined to be active (LC 90 < 20 µg/mL) [42].

Toxicity of Essential Oils to the Non-Target Anisops Bouvieri
The essential oils exhibited similar trends in toxicity to A. bouvieri to the larvae of mosquito species (Table 13). The 90% lethal dose at 24 h of PG03 and PG05 essential oils for Ae. aegypti, Ae. albopictus, and Cx. fuscocephala were 1.75, 1.10, 6.40 and 0.68, 0.84, 6.12 µg/mL, respectively.

Essential Oil Chemotypes
There are many different cultivars of P. guajava, and the volatile phytochemical profiles have shown wide variation. In order to place the six cultivars from Vietnam into a phytochemical context, a hierarchical cluster analysis (HCA) comparing the major components of 120 essential oils that were reported in the literature from 2015 to 2023, refs.  as well as the six specimens from Vietnam, was carried out (Figure 1). The cluster analysis revealed at least eight clusters. Cluster 1 is characterized by a high content of limonene and (E)-β-caryophyllene and contains two Vietnamese samples, PG02 and PG05. Cluster 2 is a group of (E)-β-caryophyllene, and essential oils PG01, PG03, PG04, and PG06 fall into this group. Cluster 3 is a group of components α-humulene, (E)-caryophyllene and is followed by selin-11-en-4α-ol. Cluster 4 is a group of components (E)-caryophyllene, α-selinene, and 14-hydroxy-9-epi-(E)-caryophyllene. Cluster 5 is a group of (E)-β-ocimene. Cluster 6 is a group of β-bisabolol and α-humulene. Cluster 7, made up of a single sample, is dominated by (E)-nerolidol. Finally, Cluster 8 is a group containing low concentrations of (E)-nerolidol.
Mixtures of the main components in their respective proportions in the essential oils were evaluated for larvicidal activities against two species of Aedes (Tables 4 and 7). All blends have shown much weaker toxicity than their respective essential oils. These results suggested that minor components were mainly responsible for the larvicidal activities of essential oils, which may have been through synergistic effects with the major components or between the minor components [87][88][89][90][91][92][93][94][95][96]. Some scientists believe that the main components within a certain concentration range will be mainly responsible for the biological activity of the essential oils; when the threshold concentration is exceeded, the effectiveness is attributed to the synergistic interaction of/with the minor compounds [97,98]. Scalerandi and co-authors found that insects preferentially oxidize the major terpenes in the mixture, while the minor terpenes act as toxicants [99]. Interestingly, the two essential oils, PG02 and PG05, were almost identical in terms of composition and content; however, PG05 has shown several times stronger toxicity to the larvae of three mosquito species than PG02. Van Vuuren and Viljoen have found that synergistic, antagonistic, or additive effects depend on the ratio and specific enantiomer [100]). Our previous study showed that the toxicity of a mixture of main components to the larvae of different mosquito species also varies [43]. Mendes and co-authors studied the chemical composition and larvicidal activity against Ae. aegypti of five guava cultivars from Brazil, three guava cultivars that fell into Cluster 2 showed weaker activity than the three cultivars (PG01, PG04, and PG06) from Vietnam [66]. The larvicidal activities of some minor compounds (<0.5% content) in the guava cultivars' essential oils are presented in Table 14. However, none of these compounds alone can account for the strong larvicidal activities of the essential oils.  PG03 contained minor components, such as α-cadinol (LC 50-albopictus = 11.22 µg/mL) [108], α-bisabolol, epi-β-bisabolol (LC 50-aegypti = 15.83 µg/mL) [109], whereas a mixture of cadinol + α-bisabolol exhibited an LC 50-aegypti value of 2.53 µg/mL [110], suggesting that α-cadinol and α-bisabolol may be synergistic in larvicidal activities. All six essential oils contained αcopaene at concentrations above 2.0%, and it may be that these compounds that played an important role for the larvicidal activities via synergistic actions with the other components. Hymenaea courbaril fruit peel essential oil with the main components α-copaene (11.1%), spathulenol (10.1%) and β-selinene (8.2%) was effective against Ae. aegypti larvae with an LC 50 value of 14.8 µg/mL [111], while also spathulenol and β-selinene both exhibited LC 50 values > 100 µg/mL [104,107]. Callicarpa sinuata leaf essential oil contained two main components, α-copaene (12.6%) and α-humulene (24.8%), which have shown an LC 50-aegypti value of 25.86 µg/mL [112].

Molluscicidal Activities
All six P. guajava essential oils showed notable molluscicidal activities. Some of the major components in the essential oils have also shown strong molluscicidal activity against P. acuta (Table 10) and I. exustus (Table 12). Monoterpenes limonene and α-pinene have exhibited weaker toxicity than sesquiterpenoid compounds. The (E)-β-caryophyllene and maybe its synergistic actions with other constituents were responsible for the molluscicidal activity of the essential oils. Several previous studies have supported this trend. Cannabis sativa containing 18.7% of (E)-β-caryophyllene demonstrated greater toxicity to P. acuta than essential oils containing (E)-β-caryophyllene as a minor component [29,113]. (E)-Nerolidol did not exhibit toxicity against Biomphalaria glabrata at a concentration of 100 µg/mL [35]. Essential oils containing (E)-β-caryophyllene as the main constituent exhibited potent molluscicidal activities against Gyraulus convexiusculus and Pomacea canaliculata [42,43,114].
At the concentration of 1.75 µg/mL, the essential oils PG05 and PG03 were lethal to less than 1% of A. bouvieri at 48 h. Thus, the two essential oils are safe for A. bouvieri when used to control larvae of Ae. aegypti and Ae. albopictus. However, at concentrations of 6.12 and 6.4 µg/mL, nearly 90% of A. bouvieri were killed at 48 h. Similarly, other essential oils have shown non-selective toxicity to Cx. fuscocephala with SI values at 24 h between 0.7 and 1.0.
The two essential oils, PG03 and PG05, exhibited lower selective toxicities to the molluscs, P. acuta and I. exustus, with SI values at 48 h of 1.2, 0.9, and 1.74, 1.10, respectively. However, other essential oils showed less toxicity to A. bouvieri with SI values at 48 h of 2.7 to 4.6 and 3.15 to 4.51, respectively. Studies by Benelli et al. (2015) and Bedini et al. (2016) reported that the essential oils A. millefolium, H. tuberculatum, C. sativa, and H. lupulus exhibited non-selective toxicity to Cloeon dipterum when compared with the target species Cx. pipiens, P. acuta, and Ae. albopictus [29,113]. Benelli found that Carlina acaulis essential oil exhibited toxicity to Daphnia magna when compared with Cx. quinquefasciatus larvae [116]. Many previous studies have shown a tendency for essential oils to exhibit greater toxicity to A. bouvieri when compared with other non-target organisms such as Diplonychus indicus, Gambusia affinis, or Poecilia reticulata [116][117][118][119][120][121].

Plant Material
All six cultivars of guava (Psidium guajava L.) have been cultivated on a large scale in Cai Be district, Tien Giang province, Vietnam. Synthetic fertilizer NPK (synthetic N, P, and potassium; 16-16-8, w/w) was periodically applied in the months of January, April, June, and August of the year. Water has been irrigated by drip technology. Guava trees after four years of age and at two months after flowering (fruit-bearing time) were the subjects of this study. Mature leaves of six cultivars of guava were collected in October 2018 (Table 15). The collected leaves were transferred to laboratory conditions on the same day and were immediately used to extract the essential oils. According to the literature provided by the Southern Horticultural Research Institute (SOFRI) [3], Mitra and Irenaeus [122], this study contains six cultivars of guava, including 'Nu hoang' (Nữ hoàng) or 'Queen guava', 'Ruot hong da san' (Ruột hồng da sần), 'Ruot hong da lang' (Ruột hồng da láng), 'Le Dai Loan' (Lê Ðài Loan) or Taiwan guava, 'Se' (Sẻ), and 'Ruot trang' (Ruột trắng). The 'Nu hoang' cultivar is characterized by white and soft flesh, few seeds, and orbicular fruits with a diameter of about 8 cm. The 'Ruot hong da lang' or 'Pink flesh smooth skin' and 'Ruot hong da san' or 'Pink flesh rough skin' are oval fruits with an average diameter of up to 9 cm, seedy, and crunchy flesh. The 'Le Dai Loan' cultivar is introduced from Taiwan, more or less orbicular fruits, soft and white flesh, and seedy. The 'Se' cultivar is a small orbicular fruit with an average diameter of about 4 cm, pink-red and thin flesh, and seedy. This cultivar is good for making juice. The 'Ruot trang' is characterized by short oval fruit, white and soft flesh, and seedy. Pictures of the fruit, flowers, and leaves of six guava cultivars are available in the Supplementary Materials Figure S1.

Hydrodistillation
The fresh leaves were chopped and hydrodistilled with a Clevenger apparatus (Witeg Labortechnik, Wertheim, Germany) for 6 h, 70 g of material and 500 mL of distilled water per trial, and the yield was calculated as the average of four consecutive trials. The essential oils (EOs) were dried over anhydrous Na 2 SO 4 , contained in brown 5 mL-vials, and stored at 4 • C until use.

Gas Chromatographic-Mass Spectral Analysis
Each of the EOs was analyzed by GC-MS using a Shimadzu GCMS-QP2010 Ultra (Shimadzu Scientific Instruments, Columbia, MD, USA) operated in the electron impact (EI) mode (electron energy = 70 eV), scan range = 40-400 atomic mass units, scan rate = 3.0 scans/s, and GC-MS solution software. The GC column was a ZB-5ms fused silica capillary column (Phenomenex, Torrance, CA, USA) (60 m length × 0.25 mm internal diameter) with a (5% phenyl)-polymethylsiloxane stationary phase and a film thickness of 0.25 µm. The carrier gas was helium with a column head pressure of 208 kPa and a flow rate of 2.00 mL/min. The injector temperature was 260 • C, and the ion source temperature was 260 • C. The GC oven temperature program was programmed for 50 • C initial temperature; the temperature increased at a rate of 2 • C/min to 260 • C and then held at 260 • C for 5 min. A 5% w/v solution of the sample in CH 2 Cl 2 was prepared, and 0.1 µL was injected with a splitting mode (24.5:1).
Identification of the oil components was based on their retention indices determined by reference to a homologous series of n-alkanes (C 8 -C 40 ) and by comparison of their mass spectral fragmentation patterns with those reported in the databases [123][124][125][126]. The percentages of each component in the EOs are reported as raw percentages based on total ion current without standardization.

Larvicidal Biassays
Aedes aegypti and Ae. albopictus have been continuously maintained in the laboratory of Duy Tan University. The adults were fed on 10% sucrose solution and blood-fed from white mice. Egg rafts of Culex fuscocephala were collected from rice fields in Hoa Vang, Da Nang (16 • 00 49 N, 108 • 06 12 E). Each egg raft was hatched separately in plastic trays containing tap water overnight; the 3rd instar and 4th early instar larvae were used for classification based on morphological characteristics [127,128]. The larvae that survived the trial were reared to adulthood and reclassified to confirm the initial identification. All larvae were fed on a mixture of dog food and yeast at a ratio of 3:1 (w/w). All developmental stages of mosquitos were maintained at 25 ± 2 • C, 65-75% relative humidity, and a 12:12 h light/dark cycle. The 3rd instar and 4th early instar larvae were used to evaluate the larvicidal activities of essential oils and purified compounds.
Tests for larvicidal activity were performed according to two protocols as described below. All tests were performed under laboratory conditions at 25 ± 2 • C, 65-75% relative humidity, and 12:12 h light/dark cycle. Dimethyl sulfoxide (DMSO, Sigma-Aldrich, Saint Louis, MO, USA) was used to prepare 1% (w/v) stock solutions and was also used as a negative control, and pyrethrin (Sigma-Aldrich) was used as a positive control. In both protocols, the solution level in the test beakers was always within the range of 5 to 10 cm [129].
Protocol 2: This protocol was performed in the same way as protocol 1, but the test solution volume was 50 mL in 150 mL beakers instead of 150 mL in 250 mL beakers, used for essential oils, major components, and major component mixtures. Testing of each pure compound and each major component mixture was performed twice on two different days, each time using a freshly prepared stock solution. After the conclusion of the two trials, the one with the stronger toxicity result was used to analyze the results.

Molluscicidal Bioassays
The adult snails of P. acuta (approximately 10 mm in length) were collected from aquarium cement tanks in Da Nang. The snails were acclimatized to laboratory conditions (25 ± 2 • C; 70 ± 5% RH, natural photoperiod) in a glass tank (50 cm wide, 100 cm long, 30 cm water level) for 48 h before testing and fed on the leaves of Lactuca sativa L.
Five adults were randomly selected and transferred to 200 mL beakers filled with 195 mL of double distilled water. Snails were exposed to essential oils at concentrations of 50, 25, 12.5, 6.25, and 3.125 µg/mL, replicated four times each. To prevent the snails from escaping, plastic Petri dishes were used to cover the top of the test beakers. Snails were allowed to recover after 24 h of exposure by transferring them to beakers containing only 195 mL of double-distilled water and identifying dead snails after the next 24 and 48 h. Snails were considered dead when there was no sign of a contraction response when probed with a needle [29,43,113]. Copper sulfate (Xilong Chemicals, Shantou, China) was used as a positive control in this experiment [43].

Toxicity on Anisops Bouvieri
Adults of A. bouvieri (Notonectidae) were collected from the wild in Da Nang city (16 • 00 22 N; 108 • 15 45 E) with a soft mesh and were maintained in glass tanks (60 cm long × 50 cm wide) at laboratory conditions (25 ± 2 • C, 65-75% relative humidity, 12:12 h light/dark cycle) for 48 h for familiarization before testing. The adults of A. bouvieri were identified based on morphology as described by Nieser [130], Ehamalar and Chandra [131]. Evaluation of the toxicity of essential oils against A. bouvieri was performed similar to protocol 1, using 20 adults for each repetition; concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 µg/mL were used. The tests were performed under laboratory conditions at 25 ± 2 • C, 65-75% relative humidity, 12:12 h light/dark cycle. The Selectivity Index (SI) between the target organism and the non-target organism was calculated using the formula [

Conclusions
Psidium guajava is well known for its use as a source of fruit as well as medicinal benefits. In this work, the potential mosquito larvicidal activities and molluscicidal activities of essential oils from six cultivars of P. guajava growing in Vietnam have been explored. Two cultivars belong to a limonene/β-caryophyllene chemotype, while the other four were of a β-caryophyllene-rich chemotype. Two essential oils showed remarkable larvicidal activity, while all six essential oils were actively molluscicidal. The biological activities of the major components do not explain the activities of the essential oils, and the synergistic effects of minor components are likely responsible. Unfortunately, there are not enough data yet to parse out these effects. Additional research is needed to examine other chemotypes of P. guajava, seasonal and geographical variations in essential oil composition, and additional screening of essential oil components. It may be that with additional compositional data along with bioactivity data, a machine-learning approach may provide some insight into the synergistic effects of the essential oil components. Nevertheless, this study has demonstrated the larvicidal and molluscicidal potential of renewable P. guajava leaf essential oils.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12152888/s1, Figure S1: Photos of leaves, flowers, and fruits of six cultivars of guava in Vietnam; Table S1: Chemical composition of essential oils of six cultivars of guava in Vietnam.